Spectrin, an alpha/beta heterodimer, is a calcium and calmodulin-binding membrane skeleton protein. It is suspected that spectrin is involved in the formation of distinct membrane domains at the synapse and plays a role in the regulation of synaptic functions, such as, neurotransmission and release of neurotransmitters. To identify the presynaptic or postsynaptic density proteins that interact with spectrin in a domain- specific manner, spectrin and associated proteins were extracted under mild conditions from the synaptosomal fractions, negatively stained or sprayed on mica chips in glycerol and analyzed by electron microscopy. Actin filaments and other so far unidentified globular proteins were found to be associated with the 200 nm spectrin tetramers or oligomers. When the lysed hippocampal neurons or synaptosomal fractions from rat brain were treated with heterobifunctional sulfhydral cross-linking reagents, analyses of the Triton X-100, SDS or SDS and mercaptoethanol soluble fractions showed that spectrin, and the proteins tentatively identified as actin, tubulin, N-CAM and several unidentified membrane proteins were cross-linked although direct cross-linking of any of these proteins with spectrin has yet to be demonstrated. When the hippocampal neuron primary cultures were analyzed by using double immunofluorescence techniques with 4 different anti-spectrin and anti-syntaxin or SNAP-25 antibodies, spectrin was localized underneath the plasma membrane and at the synaptic densities, but spectrin did not show colocalization with the presynaptic syntaxin or SNAP-25 suggesting that these proteins are not directly associated with spectrin. Identification of the proteins with which spectrin directly associates at the synapse will help to understand the role of spectrin in synaptic functions.